Alzheimer’s & Dementia: Translational Research & Clinical Interventions 5 (2019) 597-609 Featured Article The Sant Pau Initiative on Neurodegeneration (SPIN) cohort: A data set for biomarker discovery and validation in neurodegenerative disorders Daniel Alcoleaa,b, Jordi Clarim ona,b, Marıa Carmona-Iraguia,b,c, Ignacio Illan-Galaa,b, Estrella Morenas-Rodrıgueza,b, Isabel Barroetaa,b, Roser Ribosa-Noguea,b, Isabel Salaa,b, M. Belen Sanchez-Saudin osa,b, Laura Videlaa,b,c, Andrea Subiranaa,b, Bessy Benejama,b,c, Sılvia Valldeneu , Susana Fernandeza,b,c, Teresa Estellesa,b, Miren Altunaa,b, a,b Miguel Santos-Santosa,b, Lıdia Garcıa-Losadaa,b, Alexandre Bejanina,b, Jordi Peguerolesa,b, Vıctor Montala,b, Eduard Vilaplanaa,b, Olivia Belbina,b, Oriol Dols-Icardoa,b, S onia Sirisia,b, a,b a,b Marta Querol-Vilaseca , Laura Cervera-Carles , Laia Mu~ a,b noz , Ra ul N u~ neza,b, Soraya Torresa,b, M. Valle Camachod, Ignasi Carri od, Sandra Gimeneze, Constance Delabya,f, Ricard Rojas-Garciag,h, Janina Turon-Sansg,h, Javier Pagonabarragab,i, Amanda Jimenezj, Rafael Blesaa,b, Juan Forteaa,b,c, Alberto Lle oa,b,* a Department of Neurology, Sant Pau Memory Unit, Hospital de la Santa Creu i Sant Pau – IIB Sant Pau, Barcelona, Spain b Centro de Investigacion Biomedica en Red en Enfermedades Neurodegenerativas, Ciberned, Spain c Barcelona Down Medical Center, Fundacio Catalana Sındrome de Down, Barcelona, Spain d Nuclear Medicine Department, Institut d’Investigacions Biomediques Sant Pau - Hospital de Sant Pau, Universitat Autonoma de Barcelona, Barcelona, Spain e Respiratory Department, Multidisciplinary Sleep Unit, Hospital de la Santa Creu i Sant Pau – IIB Sant Pau, Barcelona, Spain f Universite de Montpellier, CHU de Montpellier, Laboratoire de Biochimie-Proteomique clinique, INSERM U1183, Montpellier, France g Department of Neurology, Neuromuscular Diseases Unit, MND Clinic, Hospital de la Santa Creu i Sant Pau, Biomedical Research Institute Sant Pau, Universitat Autonoma de Barcelona, Barcelona, Spain h Centro de Investigacion Biomedica en Red en Enfermedades Raras, Ciberer, Spain i Department of Neurology, Movement Disorders Unit, Hospital de la Santa Creu i Sant Pau – IIB Sant Pau, Barcelona, Spain j Endocrinology and Diabetes Department, Obesity Unit, Hospital Clinic de Barcelona - IDIBAPS, Barcelona, Spain Abstract Introduction: The SPIN (Sant Pau Initiative on Neurodegeneration) cohort is a multimodal biomarker platform designed for neurodegenerative disease research following an integrative approach. Methods: Participants of the SPIN cohort provide informed consent to donate blood and cerebrospi- nal fluid samples, receive detailed neurological and neuropsychological evaluations, and undergo a structural 3T brain MRI scan. A subset also undergoes other functional or imaging studies (video-pol- ysomnogram, 18F-fluorodeoxyglucose PET, amyloid PET, Tau PET). Participants are followed annu- ally for a minimum of 4 years, with repeated cerebrospinal fluid collection and imaging studies performed every other year, and brain donation is encouraged. Results: The integration of clinical, neuropsychological, genetic, biochemical, imaging, and neuro- pathological information and the harmonization of protocols under the same umbrella allows the dis- covery and validation of key biomarkers across several neurodegenerative diseases. Discussion: We describe our particular 10-year experience and how different research projects were unified under an umbrella biomarker program, which might be of help to other research teams pur- suing similar approaches. The authors have no competing interests to declare that are relevant for *Corresponding author. Tel.: 134935565986; Fax: 134935568604. this article. E-mail address: alleo@santpau.cat https://doi.org/10.1016/j.trci.2019.09.005 2352-8737/ Ó 2019 The Authors. Published by Elsevier Inc. on behalf of the Alzheimer’s Association. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 598 D. Alcolea et al. / Alzheimer’s & Dementia: Translational Research & Clinical Interventions 5 (2019) 597-609 Ó 2019 The Authors. Published by Elsevier Inc. on behalf of the Alzheimer’s Association. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/ 4.0/). Keywords: Biomarkers; Neuroimaging; Alzheimer’s disease; Frontontemporal dementia; Dementia with Lewy bodies; Neu- rodegeneration 1. Background where results are discussed, diagnosis is disclosed, and an appropriate treatment is prescribed, if necessary. Patients Biomarkers have revolutionized our conceptualization of and/or caregivers, usually receive an appointment with a neurodegenerative diseases. In recent years, advances in the nurse to enhance the understanding of treatment posology field of biomarkers have been instrumental in understanding and eventually to clarify other medical, legal, or social as- the neurobiology underlying these disorders. Currently, bio- pects related to the disease. markers are essential to define the long preclinical stages, to As shown in Fig. 1, once the standard care has been achieve early diagnosis, to improve the selection of partici- ensured, most patients are invited to participate in research, pants in clinical trials and to monitor the effect of drugs tar- either by enrolling in clinical trials involving new investiga- geted to specific pathological pathways [1,2]. In the Sant Pau tional drugs or in any of our observational research studies. Memory Unit, as in many other teams specialized in Clinical trials are managed by a specific research team neurodegenerative diseases, we have progressively adapted composed of neurologists, neuropsychologists, a research our clinical routine and research programs to incorporate nurse, and a data manager. This team reviews inclusion/ the different biomarkers that are developed in the field. In exclusion criteria and organizes follow-up appointments this article, we describe our particular experience with this and drug administration according to each specific trial pro- process and how the different research projects were tocol. Participants of our observational studies, on the other unified under an umbrella biomarker program, which hand, may receive ancillary biomarker and/or imaging tests might be of help to other research teams pursuing similar and a specialized additional visit where supplementary ques- approaches. tionnaires and cognitive/behavioral scales are administered. The Sant Pau Memory Unit is part of the Neurology Our observational research studies include different neuro- Department at the Hospital de la Santa Creu i Sant Pau, a ter- degenerative conditions and were recently reorganized un- tiary center that offers medical care to an area in Barcelona der a global umbrella study named SPIN (Sant Pau of approximately 400,000 inhabitants. In addition, the Unit Initiative on Neurodegeneration). is a referral center for complex or atypical cases around the Catalonia region. The Sant Pau Memory Unit attends pa- tients with cognitive or behavioral symptoms referred either 2. Methods by their primary care physician or by other neurologists to 2.1. The SPIN cohort: General protocol receive specialized diagnosis and treatment and/or to facili- tate their participation in research protocols. The SPIN cohort study was launched in 2011 as an um- Patients receive a standard initial medical visit by one of brella program with the aim of grouping individual clinical our faculty or fellow neurologists. Our initial visit consists of observational studies performed in the Sant Pau Memory an extensive medical history revision, physical examination, Unit. The SPIN cohort currently includes cognitively normal and the administration of brief tests and questionnaires to participants, patients with subjective cognitive decline, mild assess cognition and functional impact in daily living activ- cognitive impairment, mild AD dementia, frontotemporal ities. In most cases, in particular when cognitive impairment lobar degeneration–related syndromes (FTLD-S), dementia is not evident, a formal 1-hour cognitive evaluation by a with Lewy bodies (DLB) and Down syndrome (which is neuropsychologist is scheduled, and sometimes, especially treated in a separate section; see DABNI project). Inclusion in patients with a suspected frontotemporal lobar and exclusion criteria for the SPIN cohort are detailed in degeneration–related syndrome or an atypical Alzheimer’s Table 1. Patients with FTLD-S are systematically referred disease (AD) syndrome, a specialized 2-hour additional visit to the Motor Neuron Disease Clinic at the Neuromuscular helps to refine their clinical diagnosis. Neuroimaging and Diseases Unit and evaluated by neurologists experienced blood tests are also usually scheduled if they have not in diagnosis and management of motor neuron diseases. A been previously ordered by their referral physician. In standardized visit is performed focusing on the detection 2009, the clinical protocol was refined and cerebrospinal of signs and symptoms suggestive of upper and lower motor fluid (CSF) biomarkers were integrated to improve the early neuron involvement. In these patients, an electrophysiolog- detection of prodromal AD. This approach facilitates the im- ical evaluation, including electromyographic concentric plementation of disease-modifying drugs that are currently needle examination, motor and sensory nerve conduction under investigation in phase 2 or phase 3 clinical trials. After studies are performed by qualified physicians according to the completion of these tests, a follow-up visit is scheduled established standards. D. Alcolea et al. / Alzheimer’s & Dementia: Translational Research & Clinical Interventions 5 (2019) 597-609 599 Primary care physician Neurologists Sant Pau Memory Unit • Extensive medical history revision Inial medical visit • Physical examinaon • Brief tests to assess cognion and Standard neuropsychological evaluaon funconal impact in daily living acvies Neuroimaging: brain scan or MRI General blood tests Lumbar puncture for CSF biomarkers Supplementary clinical and neuropsychological protocol if necessary Follow-up visit • Results discussion • Diagnosis disclosure Nurse appointment • Prescripon of appropriate treatment Are you interested in our research programs? No Yes Pharmacological clinical trials Clinical trials team Observaonal research studies SPIN cohort protocol Roune clinical follow-up Fig. 1. Flow-chart of clinical practice and research protocols in the Sant Pau Memory Unit. As the primary objective of the SPIN cohort is biomarker On the first day, a neurologist and a neuropsychologist discovery and validation, consent for blood and CSF collec- perform a full medical history review, physical examina- tion is required for all participants. Detailed neurological tion, and a standard neuropsychological evaluation and neuropsychological evaluation and a structural 3T brain (Table 2) followed by a lumbar puncture and blood extrac- MRI are also necessary for inclusion. As shown in Fig. 2, a tion. Neuroimaging studies might be scheduled on subset of participants receive a video-polysomnogram and/ different days. or imaging tests that involve radiotracers, such as 18F-fluoro- To minimize the impact of the study on volunteers’ deoxyglucose (18FDG), amyloid or Tau PET. Participants routine, all participants can choose how they prefer to be are followed on an annual basis for a minimum of 4 years, informed of the results, either by post, email, or by sched- and we offer the possibility to repeat imaging and/or CSF uling an extra on-site appointment. Those participants studies every two years. whose neuropsychological evaluation, biochemical analysis, or neuroimaging studies show clinically relevant abnormal 2.2. Cognitively normal volunteers results are scheduled for an on-site consultation to discuss these results. Cognitively normal participants are contacted Asymptomatic volunteers are an extremely relevant annually for a minimum of 4 years, and they are invited to group in the SPIN cohort. Volunteers are usually spouses repeat neuropsychological, imaging, and CSF studies every or children of patients that are informed about our studies two years. at the outpatient clinics of the Sant Pau Memory Unit. Vol- unteers can also learn about our projects through talks, our 2.3. Ethical aspects website (https://santpaumemoryunit.com) or social media (@SantPauMemory). At the moment of inclusion in our observational All volunteers receive an initial consultation with a studies, the details of the protocol are explained, and ver- neurologist in which the SPIN protocol is explained in detail, bal and signed informed consent is obtained from all par- inclusion/exclusion criteria are reviewed and informed con- ticipants. We specifically ask all participants for their sent is signed. As in other observational studies, the SPIN consent to the acquisition, analysis, and storage of biolog- cohort has a blinded design and biomarker results are not ical samples. They are also informed about the possibility disclosed to volunteers unless clinically relevant or in case of sharing anonymized information and/or biological sam- the participant becomes eligible for pharmacological pre- ples with other researchers, which is requested in an inde- vention clinical trials. pendent consent form. The original protocol and the 600 D. Alcolea et al. / Alzheimer’s & Dementia: Translational Research & Clinical Interventions 5 (2019) 597-609 Table 1 Inclusion and exclusion criteria for the SPIN cohort Inclusion criteria For all participants 18 years or older Signed informed consent Cognitively normal controls No memory complaints MMSE [27–30] CDR global score 5 0 FCSRT total immediate score (EAS62)  7 Absence of significant impairment in other domains or in daily living activities. Subjective cognitive decline Memory complaints (severe enough to have resulted in a request for medical referral) MMSE [27–30] CDR global score 5 0 FCSRT total immediate score (EAS62)  7 Absence of significant impairment in other domains or in daily living activities. Prodromal AD MMSE [24–30] CDR global score 5 0.5 Absence of a clinical diagnosis of dementia CSF biomarkers supporting AD pathophysiology Typical AD dementia CDR global score  0.5 FCSRT total immediate score (EAS62)  6 Clinical criteria of “probable AD dementia with evidence of the AD pathophysiological process” [59] Dementia with Lewy bodies Lewy body dementia: probable Lewy body dementia [60] or Lewy body mild cognitive impairment: Mild cognitive impairment [61] AND one or more of: - Visual hallucinations - Parkinsonism - REM sleep behavior disorder - Cognitive fluctuations [62] Frontotemporal lobar degeneration–related syndromes (FTLD-S) Possible, probable, or definitive behavioral variant of frontotemporal dementia [63] Semantic variant of primary progressive aphasia [64] Nonfluent/agrammatic variant of primary progressive aphasia [64] Corticobasal syndrome [65] Progressive supranuclear palsy syndromes [66,67] Any of the clinical diagnoses along the amyotrophic lateral sclerosis–frontotemporal dementia (ALS-FTD) continuum [68,69] Down syndrome Presence of trisomy at chromosome 21 Exclusion criteria  Inability to complete neuropsychological tests and questionnaires (illiteracy, blindness, hearing impairment)  Contraindication for MRI (claustrophobia, pacemaker, aneurism clips, cardiac mechanical valve)  Contraindication for lumbar puncture (anticoagulation, coagulation disease): Must not be taking anticoagulant treatment such as acenocoumarol, heparin, warfarin, dabigatran, rivaroxaban, apixaban  Current treatment with drugs that can impair cognition  Medical history of: B Neurological disease (stroke, brain lesions, epilepsy) B Psychiatric disease (psychosis or major depression) B Drug abuse in the last year B Medical history of cancer is an exclusion criterion when: - It affects the central nervous system - It has not been in complete remission for 5 years or longer - Patient has received potentially neurotoxic chemotherapy - Patient has received cranial radiotherapy Abbreviations: AD, Alzheimer’s disease; CSF, cerebrospinal fluid; EAS62, education-adjusted score at the age of 62 years [70]; MMSE, Mini–Mental State Examination; CDR, Clinical Dementia Rating; FCSRT, Free and Cued Selective Reminding Test; SPIN, Sant Pau Initiative on Neurodegeneration. D. Alcolea et al. / Alzheimer’s & Dementia: Translational Research & Clinical Interventions 5 (2019) 597-609 601 SPIN SPIN cohort protocol cohort protocol BASELINE BASELINE YEAR YEAR 11 YEAR YEAR 22 YEAR YEAR 33 YEAR YEAR 44 Revision of inclusion and exclusion criteria Required - - - - Protocol revision and general consent signature Required - - - - Medical history revision Required Update Update Update Update Clinical examinaon Required - Required - Required Neuropsychological evaluaon Required Required Required - Required General blood test Required Required Required - Required Blood extracon for plasma/serum/genecs Required - Oponal - Oponal Lumbar puncture for CSF biomarkers Required - Oponal - Oponal Brain MRI Required - Oponal - Oponal 18F-Fluorodeoxyglucose PET Oponal - Oponal - Oponal Amyloid PET Oponal - Oponal - Oponal Tau PET Oponal - Oponal - Oponal Sleep evaluaon and real world evidence data Oponal - Oponal - Oponal Fig. 2. General protocol in the SPIN cohort. Abbreviations: SPIN, Sant Pau Initiative on Neurodegeneration. subsequent amendments were approved by our local 2.5. Cerebrospinal fluid ethics committee. The SPIN cohort is based on blinded enrollment and only clinically relevant biomarker results Lumbar puncture for CSF sampling is required for all par- are disclosed. ticipants of the SPIN cohort. CSF is collected and processed in polypropylene tubes following international recommen- dations [4,5]. The first 2 ml of CSF are transferred to the 2.4. Neuropsychological evaluation general laboratory for cell count, and analysis of glucose and protein levels. Another volume of 15-20 ml is All participants in the SPIN cohort receive a standard transferred to our laboratory where samples are processed one-hour neuropsychological evaluation to assess episodic and aliquoted within the first two hours after the lumbar verbal memory, visual memory, attention, executive func- puncture. Aliquots are stored at 280 C until analysis. Full tions, visuospatial, visuoperceptive and visuoconstructive protocol for CSF processing is detailed in Fig. 3A. functioning and language. Neuropsychiatric symptoms, Core AD biomarkers (Ab1-42, t-Tau, and p-Tau) are functional impact, and the level of global cognitive impair- routinely measured in all participants. Our clinical cutoffs ment are also assessed. Table 2 lists all tests included in for core AD biomarkers were initially obtained from a group our standard neuropsychological evaluation [3]. of 70 patients clinically diagnosed with dementia of the Alz- Our standard protocol includes two verbal memory tests heimer type (whose clinical diagnoses were made blind to that are sequentially administered to maximize the detection biomarker results) and 45 age-matched cognitively normal of prodromal AD: the Free and Cued Selective Reminding controls. Our internal cutoffs were calculated using ELISA Test (FCSRT) and, after a nonverbal interference task, the [6], transferred to fully automated platforms and validated Consortium to Establish a Registry for Alzheimer’s Disease in a sample of patients that underwent amyloid PET [7]. (CERAD) word list when the former is within normal range. Our laboratory participates in the Alzheimer’s Association We have found that the combination of these two tests can quality control program for CSF biomarkers [8,9]. improve the early detection of AD and define prognostic pro- files in mild cognitive impairment [3]. 2.6. Blood and DNA extraction Participants with a diagnosis of DLB or FTLD-S receive an additional neuropsychological evaluation to further All participants in the SPIN cohort have blood drawn at investigate cognitive functions that are particularly impaired the time of lumbar puncture. Fasting is not required before in these disorders, such as visuoperceptive functions and the extraction, but the time from last meal to blood extrac- fluctuations in DLB or language, behavior, and executive tion is recorded. All samples are transferred to our laboratory functions in FTLD-S (Table 2). where they are centrifuged and aliquoted within 2 hours after 602 D. Alcolea et al. / Alzheimer’s & Dementia: Translational Research & Clinical Interventions 5 (2019) 597-609 Table 2 Table 2 Clinical and neuropsychological evaluation protocols Clinical and neuropsychological evaluation protocols (Continued ) Standard neuropsychological evaluation Additional clinical and neuropsychological evaluation in FTLD-S patients Screening tests Extended phonemic verbal fluency test [78,79] Mini–Mental State Examination [71,72] Narrative writing of the Boston cookie theft picture [84] and free Memory Alteration Test [73] extensive narrative writing without time constraints (1 full A4 page) Episodic verbal memory* Spanish adaptation of the New Adult Reading Test named “Test de Free and Cued Selective Reminding Test (FCSRT) [70,74] acentuacion de palabras” [104] Consortium to Establish a Registry for Alzheimer’s Disease (CERAD) Attention/executive functions word list [75] Design fluency of the D-KEFS battery [105] Visual memory Stroop test [106] Complex Rey Figure Recall [70,76,77] Frontal Assessment Battery [107] Geometric Figures recall from the Consortium to Establish a Registry for Behavior and mood Alzheimer’s Disease or CERAD battery [75] Frontal Behavioral Inventory [108] Attention/executive functions Informant version of the Lille Apathy Rating Scale [109] Phonemic verbal fluency test [78,79] Cambridge Behavioral Inventory Revised [110] Trail-Making test form A and B [80] Brief questionnaire of Socioemotional Dysfunction WAIS Direct and Reverse Digit span [80] Clock Drawing Task on command [81] Abbreviations: ALS-FTD, amyotrophic lateral sclerosis–frontotemporal Language dementia; FTLD-S, frontotemporal lobar degeneration–related syndromes; Boston naming test [82,83] CATFI, Catalan Initiative for Frontotemporal Dementia; UPDRS-III, Uni- Consortium to Establish a Registry for Alzheimer’s Disease or CERAD fied Parkinson’s Disease Rating Scale, Part III (motor examination). battery (orders comprehension) [75,84] *Tests that assess episodic verbal memory are administered sequentially Semantic verbal fluency [78,79] separated by a nonverbal interference task. Visuospatial/visuopercetpive/visuoconstructional functioning Geometric figures copy subtest of CERAD Number location subtest from the Visual Object Space and Perception extraction and stored at 280 C until they need to be (VOSP) battery [85] analyzed. Full protocol for blood processing is detailed in Poppelreuter overlapping figures test [86] Fig. 3B. Neuropsychiatric symptoms DNA is extracted from whole blood samples using the Neuropsychiatric inventory [87] DNeasyÒ Blood & Tissue kit (Qiagen). APOE genotype is Geriatric depression scale [88] Functional assessment routinely determined for all participants in the SPIN cohort Interview for deterioration in daily living in dementia [89] by direct DNA sequencing of exon 4 and visual analysis of Global cognitive impairment the resulting electropherogram is performed to identify the Global deterioration scale [90] two coding polymorphisms that encode the three possible Clinical dementia rating [91] apoE isoforms. Other genetic studies in the SPIN cohort FTLD-specific Clinical Dementia Rating [92] involve mendelian genes related to neurodegenerative de- Additional neuropsychological evaluation in LBD patients mentias (i.e., PSEN1, PSEN2, APP, C9orf72, MAPT, VCP, Visual Object Space and Perception (VOSP) battery [85] etc.), as well as other genes and genomic regions that have Line Orientation Test been involved in their genetic architecture, such as TREM2 Facial Recognition Test [10], CHCHD10 [11], TUBA4A [12], or the 17q21.31 region Pareidolia Test around MAPT [13]. High-throughput genotyping and next- Clinician Assessment of Fluctuation generation sequencing technologies are also used in specific Additional clinical and neuropsychological evaluation in FTLD-S patients studies [14,15]. Standardized neurological examination (CATFI protocol) including UPDRS-III [93] Separate clinical interview with at least 1 reliable informant for a precise 2.7. Structural, diffusion, and functional MRI determination of the chronology of both cognitive and behavioral signs All participants in the SPIN cohort are required to un- and symptoms [94] Frontotemporal Dementia Rating Scale [95,96] dergo 3T-MRI for structural, diffusion, and resting-state In ALS-FTD patients: Edinburgh Cognitive and Behaviour ALS Screen functional MRI. Acquisitions parameters are detailed in (ECAS) [97,98] Table 3. Extensive language evaluation (audio recorded) Our neuroimaging core takes advantage of surface-based Progressive Aphasia Language Scale [99,100] methodologies to process MRI images. Structural MRI is Spontaneous speech and description of Boston cookie theft picture test [84] processed using FreeSurfer software package (v6.0; http:// Standardized examination of Apraxia of Speech including alternating surfer.nmr.mgh.harvard.edu) to obtain individuals’ cortical movement rate and sequential motion rate tasks [101] reconstruction and cortical thickness maps by using previ- Repetition of sentences and pseudowords [84] ously described methods [16]. Estimated surfaces are then Syntactic processing [84] inspected to detect possible errors in the automatic segmen- Pyramid palm and trees test [102,103] (Continued ) tation procedure, and manual edits are performed to guar- antee an accurate cortical segmentation. D. Alcolea et al. / Alzheimer’s & Dementia: Translational Research & Clinical Interventions 5 (2019) 597-609 603 A Lumbar puncture Local laboratory Alzheimer laboratory 1ml Aliquot 120ul (8) 1 ml CSF 2 x 10 ml CSF 3ml regular tube Sarstedt 10ml Centrifuge Aliquot 270ul (10) (green cap) Ref. 62.610.018 2000g x 10min Rest •Cell count 4ºC Aliquot 500ul (12) •Glucose •Proteins LABEL E 100 / 250 / 500ul CSF SARSTEDT 2ml REF. 72.694.007 polypropylene STORE -80ºC until analysis B Blood extraction 1 x 10ml 1 x 10ml Local Laboratory 8 x 10ml whole blood serum EDTA 1 x 10ml 4 x 10ml 1 x 10ml RNA EDTA serum 1 x 10ml 1 x 10ml 3 x 10ml 1 x 10ml Genetics Biomarkers PLASMA PELLET SERUM (EDTA) Alliquot Vortex x 10sec Alliquot and alliquot LABEL A LABEL LABEL LABEL 480ul B B B LABEL C LABEL D DNA and RNA extraction Centrifuge plasma 120 ul 270ul 500ul +20uL Prot plasma plasma plasma 500ul pellet 500ul serum inhibitor (2) (5) (8) (>10) (4) (>7) 2000g x 10min 4ºC SARSTEDT 1.5ml REF. 72.690.001 STORE -80ºC until analysis Fig. 3. Biofluid processing: protocols for cerebrospinal fluid (A) and blood (B). Diffusion images are processed with a surface-based in- visually rated by an expert in nuclear medicine, and images house developed algorithm [17,18] based on FSL (http:// are also processed and quantified by our neuroimaging team. fsl.fmrib.ox.ac.uk/fsl/fslwiki) and FreeSurfer tools. Our For the whole-brain voxelwise analysis, 18FDG PET images pipeline computes cortical diffusivity metrics that allow are intensity-scaled by the reference pons-vermis region, the evaluation of microstructural changes in the cortex, spatially normalized using SPM12 (http://www.fil.ion.ucl. mitigating some of the pitfalls caused by usual voxel- ac.uk/spm/) to the Montreal Neurological Institute PET tem- based methodologies. Resting-state functional MRI studies plate and spatially smoothed with a Gaussian kernel of full are processed with an in-house algorithm specifically devel- width at half maximum 8 mm. All resulting images are visu- oped for these sequences [19]. This pipeline admits proced- ally inspected to check for possible registration errors. The ures commonly used in functional imaging studies such as quantitative analysis of 18FDG uptake in a specific region seed analysis and independent component analysis. of interest is performed following the methodology proposed by Landau et al. [20–22]. 2.8. Nuclear medicine imaging: 18FDG PET, amyloid Over the past few years, new tracers have been developed PET, Tau PET to visualize in vivo specific neuropathological aggregates. In the SPIN cohort, amyloid PET studies with 18F-florbetapir Studies with 18FDG PET are acquired in a subset of par- have been acquired for a subset of participants, and more ticipants to measure brain metabolism. These studies are recently, we have incorporated 18F-flutemetamol and tau 604 D. Alcolea et al. / Alzheimer’s & Dementia: Translational Research & Clinical Interventions 5 (2019) 597-609 Table 3 MRI acquisition parameters for structural, diffusion, and functional MRI MRI acquisition parameter Structural T1 MRI Diffusion-weighted imaging MRI Resting-state functional MRI Protocol MPRAGE Echo-planar Imaging Echo-planar Imaging Repetition Time (ms) 8.1 13,677 200 Echo Time (ms) 3.7 61 31 Slices 160 80 21 Slice thickness (mm) 1 2 4 (1 of gap) Voxel Size (mm) 0.94!0.94!0.94 2!2!2 2.4!3.3!4 Specific Parameters - Single b 5 0 image. 32 gradient directions 190 volumes. Eyes open. with b 5 1000. One inverted phase encoding b 5 0 Abbreviation: MPRAGE, magnetization-prepared rapid acquisition with gradient echo. tracers. Images are visually rated by experts in nuclear med- be compared with subjective sleep diary reports and objec- icine, and specifically developed semiautomated quantifica- tive polysomnographic data. tion protocols are applied [7]. Briefly, amyloid PET images are spatially normalized to the subject’s T1-MRI and then to 2.10. Neuropathology the Montreal Neurological Institute 152 template using the individual’s anatomical MRI coregistration. Global amyloid All participants in the SPIN cohort are encouraged to con- PET standardized uptake value ratio scores are estimated sent for brain donation. In follow-up visits, we stress the averaging frontal, cingulate, parietal, and temporal cortices importance of neuropathological studies to advance the and using the whole cerebellum as the reference region research of neurodegenerative diseases and suggest all par- [23]. In addition to the commonly used analyses in PET us- ticipants contact the Barcelona’s Neurological Tissue Bank ing voxel-based and region-based approaches, we have (http://www.clinicbiobanc.org) for formal registration. added surface-based methodologies that substantially Neuropathological validation is a critical issue in multi- improve the reliability of PET effects and reduce the inter- modal biomarker studies. Brain donation not only allows subject variance. the confirmation of the diagnosis and the presence of comor- bid pathologies but is also necessary to correlate patholog- ical data with CSF or imaging biomarkers [24] and to 2.9. Sleep evaluation and real-world evidence data characterize the underlying biological processes of these Cognitively normal volunteers in the SPIN cohort and biomarkers [25]. Moreover, it is known that secondary pa- participants with Down syndrome are invited to undergo a thologies can influence the biomarker signatures [26,27], full sleep evaluation with subjective and objective measures and it is important to capture them for their correct of the nocturnal sleep and the circadian sleep-wake pattern. interpretation. This evaluation includes an interview with a sleep specialist, In our group, we have implemented array tomography a full nocturnal video-polysomnography, and actigraphy. microscopy, which is based on obtaining ultrathin (70 nm) Participants are requested to track their sleep-wake pattern consecutive brain sections combined with immunofluores- in sleep diaries and to answer sleep questionnaires designed cence [28,29]. Array tomography requires special fixation to assess participants’ self-reported sleep quality (Pittsburgh conditions, which limits the availability of samples for this Sleep Quality Index), somnolence (Epworth Sleepiness type of study. This technique has been previously applied Scale) and to identify participants at risk for sleep apnea syn- to successfully evidence synaptic abnormalities in AD and, drome (Berlin Questionnaire). Video-polysomnography more recently, in DLB [30–33]. studies are performed in individual, sound-attenuated, temperature-regulated sleep unit rooms under continuous 2.11. Data integration supervision of qualified technical staff. All-night video-pol- ysomnography records information from 19 electroencepha- One of the strengths of the Sant Pau Memory Unit is that lographic channels, two oculographic electrodes, four we have integrated clinical, neuropsychological, biomarker, surface electromyographic channels, and six sensors to genetic, neuroimaging, and neuropathological information monitor respiratory function by assessing oximetry, oronasal from subjects with different neurodegenerative disorders in airflow, thoracoabdominal movements, and snoring detec- one single database. Every participant receives a unique tion. The week before the polysomnography, participants code to integrate all the information. As all data are associ- wear an actigraph in the nondominant arm to monitor rest/ ated to specific time points for each participant, information activity cycles. These portable devices record data on levels from different categories can easily be combined, and auto- of daily activity and light during 24 hours. They provide in- mated ready-to-analyze anonymized datasheets can be ob- formation about total sleep time and sleep efficiency that can tained. D. Alcolea et al. / Alzheimer’s & Dementia: Translational Research & Clinical Interventions 5 (2019) 597-609 605 2.12. The Down-Alzheimer Barcelona Neuroimaging The incorporation of sleep evaluations in some of the Initiative: DABNI SPIN cohort clinical groups has yielded clinically relevant results. For example, in participants with Down syndrome, The Alzheimer-Down Unit was founded in 2014 as an questionnaires do not completely reflect sleep disturbances alliance between the Sant Pau Memory Unit and the Down detected on polysomnography, and data acquired by actigra- Syndrome Catalan Foundation. The specific purpose of phy might be more sensitive to detect daytime sleep this Unit, the first of its kind in the world, is the assessment compared with self-reported scales [58]. of adults with Down syndrome by medical professionals Access to brain tissue in subjects with antemortem specialized in dementia for the detection of AD. This multi- imaging or with biofluid biomarkers also offers the possibil- disciplinary Unit, formed by neurologists, neuropsycholo- ity to look into innovative clinical-pathological correlations. gists and social workers, has been recognized by the The addition of a sophisticated neuropathological quantita- Catalan government as the reference hospital in Catalonia tive technique such as array tomography microscopy to a for the assessment of neurological disorders associated multimodal biomarker program has opened the possibility with Down syndrome. of correlating biomarkers with detailed neuropathological Taking advantage of the SPIN cohort structure, the traits. As an example, in the future, we could correlate amy- Alzheimer-Down Unit launched a parallel comprehensive loid load measured by amyloid PET with synaptic densities biomarker study entitled “Down-Alzheimer Barcelona Neu- in different brain regions or a synaptic protein in CSF with roimaging Initiative” (DABNI). The aim of the DABNI proj- synaptic densities postmortem. ect is to improve our understanding of the mechanisms that drive AD in Down syndrome (http://fcsd.org/). 4. Discussion The organization of the Sant Pau Memory Unit is in many 3. Results aspects similar to other units across the globe. However, there Our database includes neuropsychological data of more are some key aspects that are specific to our Unit and that than 6000 participants, genetics data of more than 3200 par- may have been instrumental in achieving certain objectives. ticipants, more than 2600 blood samples from 2100 partici- First, the current size of our unit (around 35 members) al- pants, and more than 2000 CSF samples from 1700 lows daily interaction and collaboration under a manageable participants. Neuroimaging information of over 1100 MRI environment. Larger organizations tend to divide into studies, more than 800 FDG-PET and 200 amyloid PET smaller groups, hampering daily interaction and limiting are also incorporated and matched to clinical, neuropsycho- the possibilities of collaboration. Also, the research cores, logical, genetic, and biomarker information. such as genetics, biofluid, imaging, or neuropathology, are CSF samples from the SPIN cohort have contributed to developed inside the Unit, which facilitates the flow of pro- several international multicenter CSF biomarker studies jects and collaborations. Second, our projects are performed [34–38]. The SPIN cohort has contributed to the in a highly multidisciplinary environment that facilitates characterization of different CSF biomarkers such as b-site cross-fertilization and the generation of new ideas. Third, APP-cleaving enzyme activity [39,40], sAPP-b [6,39,41], our members hold a collaborative spirit and share leadership. neurofilament light chain [41], YKL-40 [6,39,41–43], The experience and knowledge of consolidated researchers progranulin [44], a panel of synaptic proteins [45], and mito- are complemented by fresh and innovative approaches of chondrial DNA [46] in different neurodegenerative diseases. younger researchers. Finally, our hospital environment pro- In addition, the collection of paired blood and CSF samples vides our research with a clinical patient-focused perspec- offers the possibility to investigate the correlation of tive, which helps in keeping a holistic view on biomarker levels between these two compartments [15,44]. neurodegenerative diseases. In addition, our DNA repository has been involved in The SPIN cohort is a multimodal biomarker platform de- many international studies aimed at identifying the genetic signed to study neurodegenerative diseases under a holistic basis of neurodegenerative disorders [47–50]. approach. The integration of clinical, neuropsychological, Imaging MRI studies acquired in the SPIN cohort have genetic, biochemical, imaging, and neuropathological infor- contributed to multicenter collaborative studies [17,42,51], mation and the harmonization of protocols under the same and their combination with other biomarkers has yielded umbrella has allowed the discovery and validation of key relevant results regarding multimodal relationships biomarkers for these diseases. The data generated are crucial [41,42,44]. Our neuroimaging team has also developed and to understanding the pathophysiology of neurodegenerative described novel methods for the detection of longitudinal diseases and to improve their diagnostic and prognostic changes in cortical structure [52] and cortical microstruc- assessment. tural changes in diffusion-weighted sequences in AD and Acknowledgments FTLD-S [17]. Nuclear medicine imaging studies have also contributed to several international multicenter imaging The authors thank all participants and their families for their biomarker initiatives [53–57]. generosity. They also acknowledge the Radiology 606 D. Alcolea et al. / Alzheimer’s & Dementia: Translational Research & Clinical Interventions 5 (2019) 597-609 Departments of Hospital del Mar and Hospital Clınic and the [2] Lleo A, Cavedo E, Parnetti L, Vanderstichele H, Herukka SK, Barcelona’s Neurological Tissue Bank for their collabora- Andreasen N, et al. Cerebrospinal fluid biomarkers in trials for Alz- heimer and Parkinson diseases. Nat Rev Neurol 2015;11:41–55. tion and Concepci on Escola for nursing handling. [3] Sala I, Illan-Gala I, Alcolea D, Sanchez-Saudinos MB, Salgado SA, The SPIN cohort has received funding from CIBERNED; In- Morenas-Rodrıguez E, et al. Diagnostic and prognostic value of the stituto de Salud Carlos III; jointly funded by Fondo Europeo combination of two measures of verbal memory in mild cognitive de Desarrollo Regional (FEDER), Uni on Europea, “Una impairment due to Alzheimer’s disease. J Alzheimer’s Dis 2017; manera de hacer Europa”; Generalitat de Catalunya; 58:909–18. [4] Del Campo M, Mollenhauer B, Bertolotto A, Engelborghs S, Fundaci o “La Marat o TV3” Fundaci o Bancaria Obra Social Hampel H, Simonsen AH, et al. Recommendations to standardize La Caixa; Fundaci on BBVA; Fundaci on Espa~ nola para el preanalytical confounding factors in Alzheimer’s and Parkinson’s Fomento de la Investigaci on de la Esclerosis Lateral disease cerebrospinal fluid biomarkers: an update. Biomark Med Amiotr ofica (FUNDELA); Global Brain Health Institute; 2012;6:419–30. Fundaci o Catalana Sındrome de Down; and Fundaci o Vıctor [5] Vanderstichele HMJ, Janelidze S, Demeyer L, Coart E, Stoops E, Herbst V, et al. Optimized standard operating procedures for the anal- Grıfols i Lucas. These funding sources had no role in study ysis of cerebrospinal Fluid Ab42 and the ratios of Ab isoforms using design; in the collection, analysis, and interpretation of data; low protein binding tubes. J Alzheimers Dis 2016;53:1121–32. in the writing of the report; or in the decision to submit the [6] Alcolea D, Martınez-Lage P, Sanchez-Juan P, Olazaran J, Antunez C, article for publication. Izagirre A, et al. 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